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Molecular biological method for rapidly identifying Hoolock leuconedys and Nomascus leucogenys

A molecular biology and gibbon technology, applied in the field of molecular biology, can solve the problems of identifying difficult criminals with materials, distinguishing and identifying Eastern White-browed Gibbons and Northern White-cheeked Gibbons, etc., to protect biodiversity, avoid transportation difficulties, and reduce transportation. difficult effect

Inactive Publication Date: 2017-05-10
SOUTHWEST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, relevant departments such as entry-exit animal quarantine and forest public security are facing a serious problem: how to quickly and accurately identify the fragmented tissues, organs, mutilated individuals and processed products of these rare and endangered wild animals? The reality is that in many cases due to the difficulty of material identification, the criminals cannot be punished in time
[0009] Currently, only the complete mitochondrial genome sequence (NC_021957) of the northern white-cheeked gibbon and the cytb sequence (Y13304, 1141 bp) of the western hoolock gibbon (Hoolock hoolock, western hoolock gibbon) are in Genbank, and there is no gene sequence of the eastern hoolock gibbon in Genbank. At home and abroad, there is no report on the use of COI gene fragments as molecular markers to identify eastern hooze gibbons and northern hooze gibbons

Method used

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  • Molecular biological method for rapidly identifying Hoolock leuconedys and Nomascus leucogenys
  • Molecular biological method for rapidly identifying Hoolock leuconedys and Nomascus leucogenys
  • Molecular biological method for rapidly identifying Hoolock leuconedys and Nomascus leucogenys

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Experimental program
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Embodiment 1

[0042] 1. Sample collection and storage

[0043] Four samples were used in the experiment of the present invention, including two eastern hoomo gibbons, "Bei Bei" and "Gua Gua", and two northern white-cheeked gibbons, "Le Le" and "Wei Wei". See Table 1 for sample details.

[0044] Table 1. Conditions of the samples used in the experiment of the present invention.

[0045]

[0046]

[0047] 1.1 Blood samples

[0048] The samples were collected from fresh venous blood from the eastern hoolock gibbon "Bei Bei" and the northern white-cheeked gibbon "Lele". Eastern Hoolock Gibbon "Bei Bei", female, 9 years old, was rescued by the Yunnan Provincial Wildlife Shelter and Rescue Center in Dehong Prefecture, Yunnan Province in 2015. The northern white-cheeked gibbon "Lele", female, 18 years old, was rescued by the Yunnan Provincial Wildlife Shelter and Rescue Center in Xishuangbanna Prefecture, Yunnan Province in 2006. The morphological identification of the two gibbons was co...

Embodiment 2

[0115] The object to be tested is a piece of muscle tissue, with an area of ​​about 0.3×0.4cm and a weight of about 1.6g. It was provided by the Forest Public Security Bureau of Yunnan Province. It is suspected to be a species of Eastern Hoolocks Gibbon and Northern White-cheeked Gibbon.

[0116] Take about 100 mg of the object to be tested in the sample tube, and cut the tissue pieces into pieces with clean scissors. Directly use the DNA extraction kit (Shanghai Huashun Biological Engineering Co., Ltd.) to extract. During the extraction process, a little more RNase A needs to be added to minimize RNA residue to ensure DNA quality.

[0117] The PCR amplification reaction system, PCR reaction conditions and gene sequence determination were all consistent with the samples in Example 1.

[0118] Compare the COI gene sequence of the test object directly with Table 5. For example, first look at the 10th nucleotide position, and the result shows that the base to be tested is "A"; ...

Embodiment 3

[0120] The object to be tested is a piece of fur, with an area of ​​about 1.3×1.6cm, provided by the Forest Public Security Bureau of Yunnan Province. It is suspected to be a species of eastern hooze gibbon and northern white-cheeked gibbon.

[0121] Hair DNA extraction, PCR amplification reaction system, PCR reaction conditions and gene sequence determination are all consistent with the samples in Example 1.

[0122] Compare the COI gene sequence of the test object directly with Table 5. For example, first look at the 10th nucleotide position, and the result shows that the base to be tested is "G"; secondly, look at the 42nd nucleotide position, and the base to be tested is "C". By analogy, we can finally conclude that the source of the fur tissue sample is the northern white-cheeked gibbon.

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Abstract

The invention discloses a molecular biological method for rapidly identifying Hoolock leuconedys and Nomascus leucogenys and belongs to the field of molecular biology. Genomic DNA is separated and extracted from to-be-detected objects and serves as a template; PCR is performed on the basis of a P1 primer pair and a P2 primer pair, and fragments A and B are obtained; a COI (cytochrome C oxidase I) gene complete sequence is obtained from the fragments A and B by splicing and cutting; species identification is performed according to the nucleotide sequence feature variation sites, disclosed for the first time, of the Hoolock leuconedys and the Nomascus leucogenys. Rapid and accurate molecular identification of the Hoolock leuconedys and the Nomascus leucogenys is realized for the first time, and various defects of the traditional morphological identification method are overcome. Identification objects include venous blood, feces, hair or urine samples of animals, and the method has the characteristics of being accurate, rapid and convenient, and has important function and significance for maintaining an ecosystem and protecting biodiversity.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a molecular biology method capable of rapidly distinguishing the eastern hooze gibbon and the northern white-cheeked gibbon. Background technique [0002] There are 4 genera and 17 species in the family Hylobatidae. There are 3 genera and 6 species in China. leucogenys), western black crested gibbon (N. concolor), eastern black crested gibbon (N. nasutus), Hainan gibbon (N.hainanus) and white-handed gibbon (Hylobates lar) in the genus Gibbon. [0003] The eastern hoolock gibbon and the northern white-cheeked gibbon are both distributed in Yunnan Province, China. The former is found in Dehong Prefecture and Baoshan City; the latter is found in Mengla and Lvchunhuanglian Mountains in Xishuangbanna. In recent years, due to the shrinking and changing of habitats and the frequent occurrence of crimes such as poaching and hunting, the populations of the two gibbons are in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/156
Inventor 李青青李有龙王昌命郭爱伟杨亚晋陈粉粉唐杨春冯林贵张健陈敏
Owner SOUTHWEST FORESTRY UNIVERSITY
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